CN102333862B - Differentiation of the human embryo stem cell to pancreatic endocrine pedigree - Google Patents

Differentiation of the human embryo stem cell to pancreatic endocrine pedigree Download PDF

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CN102333862B
CN102333862B CN200980153742.5A CN200980153742A CN102333862B CN 102333862 B CN102333862 B CN 102333862B CN 200980153742 A CN200980153742 A CN 200980153742A CN 102333862 B CN102333862 B CN 102333862B
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cell
expression
pedigree
characteristic markers
stem cell
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CN102333862A (en
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A·雷扎尼亚
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詹森生物科技公司
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Abstract

The present invention provides the method for promoting pluripotent stem cell differentiation.Specifically, the method with the expression of the relevant label of pancreatic endocrine pedigree is increased using TEF beta receptors activator such as activin A, activin B, activin C, GDF 8, GDF 11 or GDF15 the present invention provides a kind of.

Description

Differentiation of the human embryo stem cell to pancreatic endocrine pedigree

The priority of the application for the Serial No. 61/110,278 that application claims were submitted on October 31st, 2008.

Technical field

The present invention provides the method for promoting pluripotent stem cell differentiation.Specifically, the present invention provides one kind increase with The method of the relevant marker representation of pancreatic endocrine pedigree.

Background technology

Notice collection is caused for the progress of the cell replacement therapy of type i diabetes and the shortage of portable pancreas islet In exploitation be suitable for graft move into insulin producing cell or β cells source on.A kind of method is from how competent thin Born of the same parents, such as embryonic stem cell produce functional beta cells.

In the embryonic development of vertebrate, multipotential stem cell can produce during referred to as primitive gut embryogenesis to be included The cell colony of three germinal layers (ectoderm, mesoderm and entoderm).Such as thyroid gland, thymus gland, pancreas, intestines and liver etc Tissue will be developed via the interstage from entoderm.Interstage during this is to form definitive entoderm.It is fixed Shape endoderm cell can express a variety of labels, such as HNF-3 β, GATA4, Mixl1, CXCR4 and Sox-17.

Definitive entoderm is divided into endoderm and results in pancreas.Endoderm cells express pancreas-duodenum Hox genes Pdx1.There is no during Pdx1, pancreas is no longer developed after forming ventral pancreatic bud and dorsal pancreatic bud.Thus, Pdx1 expression Indicate a committed step in pancreas organ's generation.Except other cell types, ripe pancreas further includes outer secretion group Knit and endocrine tissue.Outer secretion and differentiation of the endocrine tissue from endoderm.

It is reported that the cell with islet cells feature is derived from the embryonic cell of mouse.For example, Lumelsky etc. People (Science 292:1389,2001) mouse embryo stem cell is reported to point of the insulin secretion structure of similar pancreas islet Change.Soria et al. (Diabetes 49:157,2000) report, the insulin secretory cell derived from mouse embryo stem cell The blood glucose in the diabetic mice of Streptozotocin induction is set to become normal.

For example, Hori et al. (PNAS 99:16105,2002) disclose, with the suppression of phosphoinositide 3-kinase (LY294002) Preparation processing mouse embryo stem cell generates the cell of similar β cells.

And for example, Blyszczuk et al. (PNAS 100:998,2003) report, from the mice embryonic of constitutive expression Pax4 Stem cell generates insulin producing cell.

Micallef et al. reports that retinoic acid adjusts embryo stem cell for directional and forms Pdx1 positive pancreatic endoderms. During the primitive gut embryogenesis end corresponding to embryo, retinoic acid is when adding in the ES cell differentiation culture of the 4th day Induce the most effective (Diabetes 54 of Pdx1:301,2005).

Miyazaki et al. reports the mice embryonic stem cell system for being overexpressed Pdx1.Theirs the results show that external source Pdx1 expression clearly enhanced in the noble cells of gained insulin, growth hormone release inhibiting hormone, glucokinase, neural element 3, Expression (the Diabetes 53 of P48, Pax6 and HNF6 gene:1030,2004).

Skoudy et al. reports that activin A (member of TGF-β superfamily) can be raised in mouse embryo stem cell The expression of pancreatic exocrine genes (p48 and amylase) and endocrine gene (Pdx1, insulin and hyperglycemic factor).Use The effect of maximum is observed during 1nM activin As.They are also observed, the expression of insulin and Pdx1 mRNA from regarding The influence of yellow acid;However, 3nM FGF7 processing causes the transcriptional level of Pdx1 to raise (Biochem.J.379:749,2004).

Shiraki et al., which have studied energy characteristic, strengthens ES cell differentiation into the growth factor of Pdx1 positive cells Effect.They observe that TGF-β 2 reproducibly produces higher proportion of Pdx1 positive cells (Genes Cells.2005 June in year;10(6):503-16).

Gordon et al. is illustrated in the case of there is no serum and pressed down there are activin together with Wnt signal transductions In the case of preparation brachyury is induced from mouse embryo stem cell+/HNF-3β+Endoderm cell (US 2006/ 0003446A1)。

Gordon et al. (PNAS, volume 103, page 16806,2006) is claimed:" Wnt and TGF-β/nodal/ activation Plain signal transduction is required for the generation institute of preceding former bar at the same time ".

However, the mouse model of embryonic stem cell development may not simulate in higher mammal (such as people) completely Development program.

Thomson et al. has separated embryonic stem cell (Science 282 from people's blastocyst:114,1998).Meanwhile Gearhart and its colleague from fetus gonad tissue derived Human embryo reproduction (hEG) cell line (Shamblott et al., Proc.Natl.Acad.Sci.USA 95:13726,1998).With can be simply by together with LIF ELISA (LIF) Cultivate to prevent the mouse embryo stem cell of differentiation different, human embryo stem cell must be maintained at it is very special under conditions of (United States Patent (USP) No.6,200,806, WO 99/20741, WO 01/51616).

D ' Amour et al. describe the generation human embryo stem cell in the presence of high concentration activator protein and low serum and spread out The enrichment culture thing (Nature Biotechnology 2005) of raw definitive entoderm.These cells are transplanted to mouse Under the scrotum, cause the more mature cell for being divided into the characteristic with some hypoblastic organses.After FGF-10 is added, people's embryo The stem cell-derived definitive endodenn cells of tire can be further differentiated into Pdx1 positive cells (US 2005/0266554A1).

D ' Amour et al. (Nature Biotechnology-24,1392-1401 (2006)) are claimed:" we have developed Go out it is a kind of by Human embryo do (hES) cell transformation into can synthesize pancreatic hormone i.e. insulin, hyperglycemic factor, growth hormone release inhibiting hormone, The differentiation method of the endocrine cell of pancreatic polypeptide and ghrelin (ghrelin).This method is by guiding cell by logical To the similar definitive entoderm for the cell that can express endocrine hormone, intestinal tube entoderm, endoderm and endocrine precursor In each stage, carry out pancreas organ in analogue body and occur.”

And for example, Fisk et al. reports the system (US2006/ for producing islet cells from human embryo stem cell 0040387A1).In this case, differentiation pathway is divided into three phases.First embryo is made one with the combination of sodium butyrate and activin A Tire stem cell is divided into entoderm.Then cell and TGF-β antagonist (such as noggin (Noggin)) are combined into EGF or β Cytokine is cultivated together, to produce Pdx1 positive cells.Terminal differentiation is induced by niacinamide.

In one example, Benvenistry et al. is claimed:" we conclude that thinking, the overexpression of Pdx1 increases The strong expression of pancreas enrichment gene (pancreatic enriched genes), the induction of insulin expression may need separately Outer exists only in internal signal (Benvenistry et al., Stem Cells 2006; 24:1923-1930)”.

Therefore, it is still obvious to need exploitation to be expanded for establishing to meet clinical needs at present, while holding is divided into again The condition of the multipotential stem cell system of the potentiality of pancreatic endocrine cell, pancreatic hormone expression cell or pancreatic hormone secretory cell. We take alternative to improve the efficiency for making human embryo stem cell break up to pancreatic endocrine cell.

The content of the invention

In one embodiment, the present invention provides the method for making pluripotent stem cell differentiation, and this method comprises the following steps:

A. multipotential stem cell is cultivated,

B. make the pluripotent stem cell differentiation into expression definitive entoderm pedigree characteristic markers cell,

C. the cell differentiation for expressing definitive entoderm pedigree characteristic markers is made into expression endoderm pedigree The cell of characteristic markers, and

D. the cell differentiation for expressing endoderm pedigree characteristic markers is made into expression pancreatic endocrine pedigree The cell of characteristic markers.

In one embodiment, it is used to increase and the relevant label of pancreatic endocrine pedigree the present invention provides one kind Expression method, the described method includes be enough to cause marker representation relevant with pancreatic endocrine pedigree to increase with comprising amount The cell of the medium treatment expression pancreatic endocrine pedigree characteristic markers of the TGF-β receptor stimulating agent added.

Brief description of the drawings

Fig. 1 shows the sketch map of differentiation scheme of the present invention.

Fig. 2 shows the expression with the relevant label of each stage of differentiation scheme of the present invention.Component a) shows Go out in the cell of the differentiated human embryonic stem cell H1 to the 1st stage by the expression of the FACS CXCR4 determined.Component B) show to determine by real-time PCR in the cell of the differentiated human embryonic stem cell H1 to the 1st stage with the 1st stage The expression of relevant label.

Fig. 3 shows differentiation to the 6th stage of differentiation scheme of the present invention and then handles the (the 7th with activin A Stage) human embryonic stem cell H1 cell dithizone dyeing.Component a) is shown before by 40 μm of cell filtering nets The difference of the cell dyed with dithizone.Component b) illustrates the ability to be dyed with dithizone by 40 μm of cell filtering nets thin The difference of born of the same parents.Component c shows to fail the difference of the cell dyed with dithizone by 40 μm of cell filtering nets.

Fig. 4 shows the method according to example 2, differentiation to the present invention use differentiation scheme the 6th stage and In the cell of the human embryonic stem cell H1 handled after breaking up to the 6th stage with activin A, determined by real-time PCR Pdx-1 (component a), nkx6-1 (component b), Pax4 (component c), nkx2.2 (component d), insulin (component e) and pancreas hyperglycaemia Plain (the expression of component f).

Embodiment

The specific embodiment part of the present invention is divided into following branch point, to describe or illustrate the present invention Some features, embodiment or application, this be for the sake of understanding disclosure, not limitation the present invention.

Definition

Stem cell is that not only self-renewing but also differentiation had produced the ability of daughter cell and define on individual cell level by them Neoblast, including self-renewing progenitor cells, non-update progenitor cells and terminal differentiation cell.The feature of stem cell also exists In:It has is divided into the functioning cell of various cell lineages by a variety of germinal layers (entoderm, mesoderm and ectoderm) in vitro And substantially contributed to most of (if not all of) after the tissue and injection blastocyst of a variety of germinal layers are produced after transplanting The ability organized the formation of.

Stem cell is divided into according to its potentiality of development:(1) it is all-round, refer to produce all embryos and extraembryonic cell class Type;(2) multipotency, refers to produce all embryonic cell types;(3) special energy, refers to produce the subgroup of cell lineage, but All cells (such as candidate stem cell (HSC) producible offspring can be produced in particular organization, organ or physiological system Cell includes:HSC (self-renewing type), be confined to the few energy progenitor cells of haemocyte and owning as blood normal components Cell type and component (such as blood platelet));(4) few energy, refers to produce the cell lineage Asia more limited than multipotential stem cell Group;And (5) single energy, refer to produce single cell pedigree (such as production of sperm stem cell).

Differentiation be non-specialization (" unoriented ") or specialization deficiency cell obtain specialized cell (such as nerve cell or Muscle cell) feature process.The cell of differentiation or the cell of induction differentiation are that more specialization has been occupied in cell lineage (" orientation ") position cell.Term " orientation " when being applied to the process of differentiation, refer in differentiation pathway into Row arrives so a kind of cell of degree:In normal circumstances, it may proceed to be divided into specific cell type or cell type Collection, and another cell type cannot be divided into normal circumstances or be returned to the relatively low cell type of differentiation degree.Dedifferente Refer to the process that cell is returned to specialization among the pedigree of cell (or orientation) degree lower ground position.As used herein, " cell Pedigree " limit cell genetic affinity, i.e., it from which cell with it what cell can be produced.Cell lineage is by cell It is positioned at the hereditary inside the plan of development and differentiation.Lineage specific markers' thing refers to special with the cell phenotype of pedigree of interest Property feature that is related and can be used in evaluating differentiation of the non-directional cell to pedigree of interest.

" beta cell pedigree " refers to there is the positive at least one of transcription factor PDX-1 and following transcription factor The cell of gene expression:NGN-3, Nkx2.2, Nkx6.1, NeuroD, Isl-1, HNF-3 β, MAFA, Pax4 and Pax6.Express β The cell of cell lineage characteristic markers includes β cells.

As used herein, the cells of definitive entoderm pedigree characteristic markers " expression " or " the 1st phase cell " or " the 1st stage " refers to the cell for expressing at least one following label:SOX-17、GATA-4、 HNF-3β、GSC、Cer1、 Nodal, FGF8, Brachyury, Mix sample homeobox protein, FGF4 CD48, it is de- in embryo protein (eomesodermin, EOMES), DKK4, FGF17, GATA-6, CXCR4, C-Kit, CD99 or OTX2.Express definitive entoderm pedigree characteristic marker The cell of thing includes former bar precursor, former bar cell, mesendodermal cell and definitive endodenn cells.

As used herein, under " cells of expression endoderm pedigree characteristic markers " refer to that expression is at least one The cell of row label:PDX-1, HNF-1 β, PTF-1 α, HNF-6 or HB9.Express endoderm pedigree characteristic markers Cell include endoderm cells, primitive gut solencyte and it is rear before enterocyte.

As used herein, the cells of pancreatic endocrine pedigree characteristic markers " expression " or " the 5th phase cell " or " the 5th stage " refers to the cell for expressing at least one following label:NGN-3、NeuroD、 Islet-1、PDX-1、NKX6.1、 Pax-4, Ngn-3 or PTF-1 α.Express pancreatic endocrine pedigree characteristic markers cell include pancreatic endocrine cell, The cell of pancreatic hormone expression cell, pancreatic hormone secretory cell and β cell lineages.

As used herein, " definitive entoderm " refers to the cell produced in primitive gut forming process from epiblast Characteristic and the cell for forming intestines and stomach and its derivative.Definitive endodenn cells express following label:HNF-3β、GATA-4、 SOX-17, Cerberus, OTX2, goosecoid, C-Kit, CD99 and Mixl1.

As used herein, " extraembryonic endoderm " refers to the cell mass for expressing at least one following label: SOX-7、AFP And SPARC.

As used herein, " label " is the nucleic acid or peptide molecule of the differential expression in cell of interest.On this Point, differential expression means the horizontal increase of positive mark's thing, and the horizontal of negative marker thing reduces.Compared with other cells, institute The detectable level of nucleic acid or polypeptide marker thing in concern cell is sufficiently high or sufficiently low so that can use this area The a variety of methods known identify cell of interest, and cell of interest and other cellular regions are mutually distinguished.

As used herein, " mesendodermal cell " refers to the cell for expressing at least one following label:CD48, it is de- in Embryo protein (EOMES), SOX-17, DKK4, HNF-3 β, GSC, FGF17, GATA-6.

As used herein, " pancreatic endocrine cell " or " pancreatic hormone expression cell " is to refer to expression at least one The cell of following hormone:Insulin, hyperglycemic factor, growth hormone release inhibiting hormone and pancreatic polypeptide.

As used herein, " endoderm cells " or " the 4th phase cell " or " the 4th stage " are to refer to expression extremely A kind of few cell of following label:NGN-3、NeuroD、Islet-1、PDX-1、PAX-4、 NKX2.2.

As used herein, " pancreatic hormone cellulation " refers to generate the cell of at least one following hormone:Pancreas islet Element, hyperglycemic factor, growth hormone release inhibiting hormone and pancreatic polypeptide.

As used herein, " pancreatic hormone secretory cell " or " the 6th phase cell " or " the 6th stage " are to refer to secrete The cell of at least one following hormone:Insulin, hyperglycemic factor, growth hormone release inhibiting hormone and pancreatic polypeptide.

As used herein, " enterocyte before rear " or " the 3rd phase cell " or " the 3rd stage " are to refer to secretion at least one The cell of the following label of kind:PDX-1、HNF-1、PTF-1A、HNF-6、HB-9、 PROX-1.

As used herein, " preceding original bar cell " refers to the cell for expressing at least one following label:Nodal or FGF8.

As used herein, " primitive gut solencyte " or " the 2nd phase cell " or " the 2nd stage " are to refer to secretion at least one The cell of the following label of kind:HNF-1 or HNF-4A.

As used herein, " former bar cell " refers to the cell for expressing at least one following label:Brachyury, Mix sample Homeobox protein or FGF4.

Separation, amplification and the culture of multipotential stem cell

The characteristic description of multipotential stem cell

Multipotential stem cell can expression phase characteristic embryonic antigen (SSEA) 3 and 4 and it is available be known as Tra-1-60 and One or more (Thomson et al., Science 282 in the label of the antibody test of Tra-1-81:1145,1998). Multipotential stem cell vitro differentiation causes the expression (if present) for losing SSEA-4, Tra-1-60 and Tra-1-81, and increases Add the expression of SSEA-1.Undifferentiated multipotential stem cell usually has alkaline phosphatase activities, which can be by using 4% poly Formaldehyde fixes cell, is then detected by the use of Vector Red as Substrate development, such as the described (Vector of manufacturer Laboratories, Burlingame Calif).Undifferentiated multipotential stem cell usually also expresses Oct-4 and TERT, this can Detected by RT-PCR.

Another desired phenotypes of the multipotential stem cell of propagation be divided into all three germinal layers i.e. entoderm, mesoderm and The potential of the cell of ectodermal histological.The versatility of multipotential stem cell can be confirmed for example in this way:By cell infusion into again In disease combined immunodeficiency (SCID) mouse, formed teratoma is fixed with 4% paraformaldehyde, group then is carried out to them Knit to examine to determine whether there is the cell type from three germinal layers.Alternatively, can be by producing embryoid body and assessing embryoid The presence with three relevant labels of germinal layer of body determines versatility.

The more of propagation can be analyzed using caryogram of the standard G with technology and the corresponding primate species announced The caryogram of energy stem cell line.Wish that there is the cell of " normal karyotype ", it means that cell is euploid, wherein owner Chromosome all exists and does not change significantly.

The source of multipotential stem cell

Multipotential stem cell is selected from human embryonic stem cell H1, H7 and H9 (WiCell) and mutant human embryonic stem cell It is BG01v (BresaGen, Athens, GA).

The culture of multipotential stem cell

In one embodiment, multipotential stem cell is usually cultivated in feeder layer, feeder cells can be in many ways Support multipotential stem cell.Alternatively, cultivating multipotential stem cell in culture systems, the culture systems are thin substantially free of raising Born of the same parents, but the same propagation for supporting multipotential stem cell is without significantly being broken up.Using by cultivating another cell class before this Type and conditioned culture medium support multipotential stem cell to be grown in the culture of no feeder cells and undifferentiated.As another One kind selection, the culture medium determined with chemical composition supporting multipotential stem cell to be grown in the culture of no feeder cells and It is undifferentiated.

For example, Reubinoff et al. (Nature Biotechnology 18:399-404 (2000)) and Thompson Et al. (Science, on November 6th, 1998:Volume 282, the 5391st phase, the 1145-1147 pages) disclose with mice embryonic into Fibroblast feeder cells layer cultivates the multipotential stem cell system from people's blastocyst.

Richards et al. (Stem Cells 21:546-556,2003) to one group of 11 kinds of different adult, fetus and Neonate's feeder layer supports that the ability of human pluripotent stem cells culture is evaluated.Richards et al. is claimed:" into The human embryonic stem cell cultivated on human skin fibroblasts feeder layer maintains human embryo stem cell form and maintains more Energy property ".

US20020072117, which discloses to produce, supports primate pluripotent stem cell in the culture of no feeder cells The cell line of the culture medium of growth.Used cell line is to obtain from embryonic tissue or come from ES cell differentiation Interstitial cell system and fibroblast-like cell system.It is thin as primary feeder that US20020072117 also discloses the cell line The purposes of born of the same parents' layer.

And for example, Wang et al. (Stem Cells 23:1221-1227,2005) disclose and exist for human pluripotent stem cells The method of long term growth in the feeder layer of derived from human embryonic stem cells.

And for example, Stojkovic et al. (Stem Cells 2,005 23:306-314,2005) one kind is disclosed to be derived from The feeder cells system of the Spontaneous Differentiation of human embryo stem cell.

In another example, Miyamoto et al. (Stem Cells 22:433-440,2004) disclose from Human plactnta The source of the feeder cells of acquisition.

Amit et al. (Biol.Reprod 68:2150-2156,2003) feeder cells of derived from human foreskin are disclosed Layer.

And for example, Inzunza et al. (Stem Cells 23:544-549,2005) disclose from people be born after foreskin into The feeder layer of fibrocyte.

US6642048, which is disclosed, can support more competent (pPS) cells of primate to be grown in the culture of no feeder cells Culture medium and available for the cell line for producing this culture medium.US6642048 is claimed:" present invention is included from embryonic tissue The interstitial cell system and fibroblast-like cell system for obtaining or coming from ES cell differentiation.Describe in the disclosure simultaneously Illustrate for deriving this cell line, processing culture medium and the method with conditioned medium cultivation stem cell ".

And for example, WO2005014799 discloses the conditioned medium for maintaining, breeding and breaking up for mammalian cell. WO2005014799 is claimed:" by mouse cell, (transgenic hepatocytes for particularly breaking up and immortalizing, are known as MMH (Met mouse Liver cell)) cell secretion activity culture medium prepared in accordance with the present invention is nursed one's health ".

And for example, Xu et al. (Stem Cells 22:972-980,2004) one kind is disclosed from derived from human embryonic stem The conditioned medium that thing obtains, the stem cell-derived thing have been subjected to genetic modification and are overexpressed human telomerase reverse transcriptase.

And for example, US20070010011 discloses a kind of culture for maintaining the chemical composition of multipotential stem cell to determine Base.

The growth factor that a kind of alternative culture systems can promote embryonic stem cell proliferation using being supplemented with without blood Clear culture medium.For example, Cheon et al. (BioReprod DOI:10.1095/biolreprod.105.046870 2005 10 The moon 19) a kind of free serum culture system of no feeder cells is disclosed, wherein embryonic stem cell, which maintains, is supplemented with and can trigger In not conditioned blood serum substituting (SR) culture medium of the different growth factors of embryonic stem cell self-renewing.

And for example, Levenstein et al. (Stem Cells 24:568-574,2006) disclosing use is supplemented with bFGF Culture medium, the method for long-term cultivation human embryo stem cell in the case of there is no fibroblast or conditioned medium.

And for example, US20050148070 is disclosed a kind of determines in serum-free and component without fibroblast feeder cells Culture medium in cultivate the method for human embryo stem cell, this method includes:Containing albumin, amino acid, vitamin, mineral Trained in the culture medium of matter, at least one transferrins or transferrins substitute, at least one insulin or insulin substitutes Stem cell is supported, which can activate fibroblast substantially without mammalian fetal serum and containing at least about 100ng/ml The supply source of the fibroblast growth factor of growth factor signal transducing receptor, the wherein growth factor is not merely into Fine cell feeder layers, the culture medium support stem cell in the case of no feeder cells or conditioned medium with undifferentiated state Propagation.

And for example, US20050233446 disclose it is a kind of be used to cultivating stem cell, including undifferentiated primate is original dry The defined medium of cell.In the solution, this culture medium is substantially isotonic compared with the stem cell in culture.What is given In culture, specific culture medium includes basal medium and is respectively a certain amount of bFGF, insulin and ascorbic acid, described BFGF, insulin and ascorbic acid is support that it is required that embryonic stem cells carry out substantially undifferentiated growth.

And for example, US6800480 is described " in one embodiment, there is provided for substantially undifferentiated state culture spirit The cell culture medium of the primordial stem cell in long class source, it includes Hyposmolality, low endotoxin basal medium, this culture medium It can be effectively used for supporting the growth of the primordial stem cell in primate source.Basal medium, which is mixed with, can effectively support primate The nutrient serum of the primordial stem cell growth in source and extracellular matrix component selected from feeder cells and derived from feeder cells Substrate.The culture medium further includes nonessential amino acid, antioxidant and the first growth factor selected from nucleosides and acetonate ".

And for example, US20050244962 is described:" in one aspect, the present invention provides one kind to cultivate primate embryonic stem The method of cell.In the culture substantially free of mammalian fetal serum (preferably also substantially free of any animal blood serum) In, and trained in the case of the fibroblast growth factor provided by the source in addition to only fibroblast feeder layer is provided Support stem cell.In a kind of preferable form, by adding enough fibroblast growth factors so that fibroblast Feeder layer (being maintained needed for stem cell culture in the past) becomes nonessential ".

In another example, WO2005065354 discloses a kind of component substantially without feeder cells and serum-free Definite isotonic culture medium, it includes:A. basal medium;B. a certain amount of bFGF, the amount are enough to support substantially undifferentiated Mammalian stem cell growth;C. a certain amount of insulin, the amount are enough to support substantially undifferentiated mammal to do carefully Intracellular growth;With a certain amount of ascorbic acid of d., which is enough to support substantially undifferentiated mammalian stem cell growth.

And for example, WO2005086845 discloses a kind of method for maintaining undifferentiated stem cell, and the described method includes make to do Cell is exposed to the member of transforming growth factor-β (TGF-β) protein family, fibroblast growth factor (FGF) albumen man The amount of the member of race or niacinamide (NIC), the member or niacinamide is enough to maintain cell to be in undifferentiated state up to being enough reality A period of time of result needed for existing.

Multipotential stem cell can be seeded on suitable culture substrate.In one embodiment, suitable culture substrate is Extracellular matrix components, such as the component from basilar memebrane that spreads out, or a part for adhesion molecule receptor-ligand couplings can be formed Component.In one embodiment, suitable culture substrate is MATRIGEL(Becton Dickenson)。MATRIGEL It is the soluble preparation derived from Engelbreth-Holm Swarm tumour cells, it is gelled and forms the base of reconstruct at room temperature Counterdie.

Other extracellular matrix components and component mixture are suitable as substitute.Depending on the cell class expanded Type, this may include single laminin, fibronectin, proteoglycans, nestin, Heparan sulfate etc. or they Various combinations.

Can be in the presence of presence can promote cell survival, propagation and keep the culture medium of ideal characterisitics, with suitable Distribution multipotential stem cell is inoculated in the matrix.All these characteristics can benefit from thinking better of inoculation distribution And can easily it be determined by those skilled in the art.

Suitable culture medium can use following component to prepare, such as Dulbecco's modified Eagle medium (DMEM), Gibco # 11965-092;Knockout Dulbeccos modified Eagle medium (KO DMEM), Gibco # 10829- 018;Ham ' s F12/50%DMEM basal mediums;200mM L- glutamine, Gibco # 15039-027;Nonessential ammonia Base acid solution, Gibco 11140-050;Beta -mercaptoethanol, Sigma # M7522;People's recombination basic fibroblast growth because Sub (bFGF), Gibco # 13256-029.

Pancreatic hormone cellulation is formed from multipotential stem cell

In one embodiment, the present invention provides a kind of method that pancreatic hormone cellulation is produced from multipotential stem cell, This method comprises the following steps:

A. the multipotential stem cell is cultivated,

B. make the pluripotent stem cell differentiation into expression definitive entoderm pedigree characteristic markers cell,

C. the cell differentiation for expressing definitive entoderm pedigree characteristic markers is made into expression endoderm pedigree The cell of characteristic markers, and

D. the cell differentiation for expressing endoderm pedigree characteristic markers is made into expression pancreatic endocrine pedigree The cell of characteristic markers.

In one aspect of the invention, pancreatic endocrine cell is pancreatic hormone cellulation.In an alternative aspect, Pancreatic endocrine cell is the cell for expressing β cell lineage characteristic markers.Express β cell lineage characteristic markers' Cell can express Pdx1 and at least one following transcription factor:NGN-3、 Nkx2.2、Nkx6.1、NeuroD、Isl-1、HNF-3 β, MAFA, Pax4 and Pax6.In one aspect of the invention, the cell for expressing β cell lineage characteristic markers is β cells.

Being suitable for the invention multipotential stem cell includes such as human embryonic stem cell H9 (NIH codings:WA09), people's embryo (NIH is encoded tire stem cell line H1:WA01), (NIH is encoded human embryonic stem cell H7:) and human embryonic stem cell WA07 SA002 (Cellartis, Sweden).The cell that at least one following peculiar label of pluripotent cell can be expressed is also applied for this hair It is bright:ABCG2, cripto, CD9, FoxD3, connection protein 43, connection albumen 45, Oct4, Sox2, Nanog, hTERT, UTF-1, ZFP42、SSEA-3、SSEA-4、Tra1-60、Tra1-81。

Definitive entoderm pedigree characteristic markers be selected from SOX-17, GATA4, Hnf-3beta, GSC, Cer1, Nodal, FGF8, Brachyury, Mix sample homeobox protein, FGF4 CD48, it is de- in embryo protein (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99 and OTX2.It is at least one definitive entoderm pedigree characteristic marker of expression to be suitable for the invention The cell of thing.In one aspect of the invention, the cell for expressing definitive entoderm pedigree characteristic markers is that former bar precursor is thin Born of the same parents.In an alternative aspect, the cell for expressing definitive entoderm pedigree characteristic markers is mesendodermal cell.At one Alternative aspect, the cell of expression definitive entoderm pedigree characteristic markers is definitive endodenn cells.

Endoderm spectrum signature label is selected from Pdx1, HNF-1 β, PTF1a, HNF-6, HB9 and PROX1.It is applicable in What it is in the present invention is the cell of expressing at least one endoderm pedigree characteristic markers.In one aspect of the invention, The cell for expressing endoderm pedigree characteristic markers is endoderm cells.

Pancreatic endocrine pedigree characteristic markers are selected from NGN-3, neurogenic differentiation albumen (NeuroD), Islet- 1st, Pdx-1, NKX6.1, Pax-4, Ngn-3 and PTF-1 α.In one embodiment, pancreatic endocrine cell can express with At least one of lower hormone:Insulin, hyperglycemic factor, growth hormone release inhibiting hormone and pancreatic polypeptide.It is table to be suitable for the invention cell Up to the cell of at least one pancreatic endocrine pedigree characteristic markers.In one aspect of the invention, pancreatic endocrine is expressed The cell of pedigree characteristic markers is pancreatic endocrine cell.Pancreatic endocrine cell can be that pancreatic hormone expression is thin Born of the same parents.Alternatively, pancreatic endocrine cell can be pancreatic hormone secretory cell.

In one aspect of the invention, pancreatic endocrine cell is the cell of expression β cell lineage characteristic markers. The cell of expression β cell lineage characteristic markers can express Pdx1 and at least one following transcription factor:NGN-3、 Nkx2.2, Nkx6.1, NeuroD, Isl-1, HNF-3 β, MAFA, Pax4 and Pax6.In one aspect of the invention, it is thin to express β The cell of born of the same parents pedigree characteristic markers is β cells.

Express the formation of the cell of definitive entoderm pedigree characteristic markers

Can by any method of this area or by any method proposed by the present invention, make pluripotent stem cell differentiation into Express the cell of definitive entoderm pedigree characteristic markers.

For example, can be according to D ' Amour et al., disclosed in Nature Biotechnology 23,1534-1541 (2005) Method, make multipotential stem cell can be divided into expression definitive entoderm pedigree characteristic markers cell.

For example, can be according to Shinozaki et al., the side disclosed in Development 131,1651-1662 (2004) Method, makes multipotential stem cell to be divided into the cell of expression definitive entoderm pedigree characteristic markers.

For example, it can be made more competent according to McLean et al., the method disclosed in Stem Cells 25,29-38 (2007) Cell can be divided into the cell of expression definitive entoderm pedigree characteristic markers.

For example, can be according to D ' Amour et al., disclosed in Nature Biotechnology 24,1392-1401 (2006) Method, make multipotential stem cell can be divided into expression definitive entoderm pedigree characteristic markers cell.

For example, it can be trained by by multipotential stem cell in the culture medium containing activin A in the absence of serum Support, then cultivate the cell together with activin A and serum, subsequently by the cell and activin A and another concentration Serum cultivate together, make pluripotent stem cell differentiation into expression definitive entoderm pedigree characteristic markers cell.This side One example of method is disclosed in Nature Biotechnology 23,1534-1541 (2005).

For example, it can be trained by by multipotential stem cell in the culture medium containing activin A in the absence of serum Support, then cultivate the cell together with activin A and the serum of another concentration, make pluripotent stem cell differentiation fixed into expression The cell of shape entoderm pedigree characteristic markers.One example of this method is in D ' Amour et al., Nature Biotechnology, disclosed in 2005.

For example, it can be not present by by multipotential stem cell in the culture medium containing activin A and Wnt ligands in serum Under cultivated, then remove and Wnt ligands and cultivate the cell together with activin A and serum, make multipotential stem cell point The cell of chemical conversion expression definitive entoderm pedigree characteristic markers.One example of this method is in Nature Disclosed in Biotechnology 24,1392-1401 (2006).

For example, can be by according to transferring LifeScan, the U.S. Patent application No.11/736 of Inc., institute be public in 908 The method processing multipotential stem cell opened, to make pluripotent stem cell differentiation for expression definitive entoderm pedigree characteristic markers' Cell.

For example, can be by according to transferring LifeScan, the U.S. Patent application No.11/779 of Inc., institute be public in 311 The method processing multipotential stem cell opened, to make pluripotent stem cell differentiation for expression definitive entoderm pedigree characteristic markers' Cell.

For example, can be by the way that according to U.S. Patent application No.60/990, the method processing disclosed in 529 be how competent thin Born of the same parents, to make cell of the pluripotent stem cell differentiation for expression definitive entoderm pedigree characteristic markers.

For example, can be by the way that according to No.61/076 is asked in United States Patent (USP), the method processing disclosed in 889 be how competent thin Born of the same parents, to make cell of the pluripotent stem cell differentiation for expression definitive entoderm pedigree characteristic markers.

For example, can be by the way that according to U.S. Patent application No.61/076, the method processing disclosed in 900 be how competent thin Born of the same parents, to make cell of the pluripotent stem cell differentiation for expression definitive entoderm pedigree characteristic markers.

For example, can be by the way that according to U.S. Patent application No.61/076, the method processing disclosed in 908 be how competent thin Born of the same parents, to make cell of the pluripotent stem cell differentiation for expression definitive entoderm pedigree characteristic markers.

For example, can be by the way that according to U.S. Patent application No.61/076, the method processing disclosed in 915 be how competent thin Born of the same parents, to make cell of the pluripotent stem cell differentiation for expression definitive entoderm pedigree characteristic markers.

Express the differentiation of the cell of definitive entoderm pedigree characteristic markers

Express the cell of definitive entoderm pedigree characteristic markers formation can by before specified scheme is carried out or The presence of the label is detected afterwards to determine.Multipotential stem cell does not express this kind of label usually.Thus, when cell starts The differentiation of pluripotent cell is detected when expressing them.

Can by by processed cell colony exposed to can specific recognition by expression definitive entoderm pedigree characteristic The reagent (such as antibody) of the protein label of the cell expression of label determines differentiation efficiency.

Method for assessing the expression of protein label and nucleic acid markers in culture or separated cell is The standard method of this area.It is in situ that these methods include quantitative reverse transcription polymerase chain reaction (RT-PCR), Northern traces Hybridization (see, for example, " Current Protocols in Molecular Biology " (Ausubel et al. (editor), 2001 Supplementary issue)) and immunoassay, such as the immunohistochemical analysis of segmentation material, western blot and for complete The flow cytometry (FACS) for the label being readily available in cell is (see, for example, " Using of Harlow and Lane Antibodies:A Laboratory Manual ", New York:Cold Spring Harbor Laboratory Press (1998))。

Multipotential stem cell is characterized in well-known to those skilled in the art, and other features of multipotential stem cell are not It is authenticated disconnectedly.Multipotential stem cell label include (such as) expression of one or more following material:ABCG2、cripto、 FoxD3、Connexin43、Connexin45、Oct4、Sox2、 Nanog、hTERT、UTF-1、ZFP42、SSEA-3、SSEA-4、 Tra1-60、Tra1-81。

, can be by the way that processed cell colony be exposed to specificity after multipotential stem cell is handled with the method for the present invention Identification by expression definitive entoderm pedigree characteristic markers cell express protein label (such as CXCR4) come into Row purifying.

Express the formation of the cell of endoderm pedigree characteristic markers

Expression definitive entoderm can be composed by any method of this area or by any method proposed by the present invention Be characteristic markers cell differentiation into expression endoderm pedigree characteristic markers cell.

For example, can be according to D ' Amour et al., disclosed in Nature Biotechnology 24,1392-1401 (2006) Method, make the cell differentiation of expression definitive entoderm pedigree characteristic markers into expression endoderm pedigree characteristic The cell of label.

At can be by using fibroblast growth factor and hedgehog signal transduction pathway inhibitor KAAD- cyclopamines The cell of reason expression definitive entoderm pedigree characteristic markers, then removes and contains fibroblast growth factor and KAAD- rings The culture medium of bar amine, and the cell is then being contained into retinoic acid, fibroblast growth factor and KAAD- cyclopamines Cultivated in culture medium, to make the cell of expression definitive entoderm pedigree characteristic markers be further differentiated into expression pancreas The cell of gland entoderm pedigree characteristic markers.One example of this method in Nature Biotechnology 24, Disclosed in 1392-1401 (2006).

In one aspect of the invention, according to transferring LifeScan, the U.S. Patent application No. 11/736 of Inc., Method disclosed in 908, by using retinoic acid and at least one fibroblast growth factor processing expression definitive entoderm spectrum Be characteristic markers cell for a period of time, to make the cell of expression definitive entoderm pedigree characteristic markers further It is divided into the cell of expression endoderm pedigree characteristic markers.

In one aspect of the invention, according to transferring LifeScan, the U.S. Patent application No. 11/779 of Inc., Method disclosed in 311, by using retinoic acid and at least one fibroblast growth factor processing expression definitive entoderm spectrum Be characteristic markers cell for a period of time, to make the cell of expression definitive entoderm pedigree characteristic markers further It is divided into the cell of expression endoderm pedigree characteristic markers.

In one aspect of the invention, by according to U.S. Patent application No.60/990, at the method disclosed in 529 The cell of reason expression definitive entoderm pedigree characteristic markers, to make expression definitive entoderm pedigree characteristic markers' Cell is further differentiated into the cell of expression endoderm pedigree characteristic markers.

Express the detection of the cell of endoderm pedigree characteristic markers

Endoderm pedigree characteristic markers are well-known to those skilled in the art, and embryo in other pancreas Layer pedigree characteristic markers are constantly authenticated.These labels can be used for whether determining cell processed according to the present invention It is differentiated and obtain endoderm pedigree features characteristic.The specific marker thing of endoderm pedigree include it is a kind of or A variety of transcription factors, such as the expression of Hlxb9, PTF-1a, PDX-1, HNF-6, HNF-1 β.

Can by by processed cell colony exposed to can specific recognition by expression endoderm pedigree characteristic The reagent (such as antibody) of the protein label of the cell expression of label determines differentiation efficiency.

Method for assessing the expression of protein label and nucleic acid markers in culture or separated cell is The standard method of this area.It is in situ that these methods include quantitative reverse transcription polymerase chain reaction (RT-PCR), Northern traces Hybridization (see, for example, " Current Protocols in Molecular Biology " (Ausubel et al. (editor), 2001 Supplementary issue)) and immunoassay, such as the immunohistochemical analysis of segmentation material, western blot and for complete The flow cytometry (FACS) for the label being readily available in cell is (see, for example, " Using of Harlow and Lane Antibodies:A Laboratory Manual ", New York:Cold Spring Harbor Laboratory Press (1998))。

Express the formation of the cell of pancreatic endocrine pedigree characteristic markers

Any method that can be by this area or any method disclosed by the invention, compose expression endoderm Be characteristic markers cell differentiation into expression pancreatic endocrine pedigree characteristic markers cell.

For example, can be according to D ' Amour et al., disclosed in Nature Biotechnology 24,1392-1401 (2006) Method, make the cell differentiation of expression endoderm pedigree characteristic markers into expression pancreatic endocrine pedigree characteristic The cell of label.

For example, can be by the cell by endoderm pedigree characteristic markers are expressed outside containing DAPT and gilamonster Secrete and cultivated in the culture medium of peptide -4, then remove the culture medium containing DAPT and exendin-4, and then by described in Cell is cultivated in the culture medium containing Exendin 1, IGF-1 and HGF, to make expression endoderm pedigree special The cell of sign property label is further differentiated into the cell of expression pancreatic endocrine pedigree characteristic markers.This method One example is disclosed in Nature Biotechnology 24,1392-1401 (2006).

For example, can be by the way that the cell for expressing endoderm pedigree characteristic markers be being contained exenatide Cultivated in culture medium, then remove the culture medium for containing exenatide, and then by the cell containing poisonous Cultivated in the culture medium of lizard excretion peptide 1, IGF-1 and HGF, to make expression endoderm pedigree characteristic markers' Cell is further differentiated into the cell of expression pancreatic endocrine pedigree characteristic markers.One example of this method is in D ' Disclosed in Amour et al., NatureBiotechnology, 2006.

For example, can be by the cell by endoderm pedigree characteristic markers are expressed outside containing DAPT and gilamonster Secrete and cultivated in the culture medium of peptide 4, be further differentiated into the cell of expression endoderm pedigree characteristic markers Express the cell of pancreatic endocrine pedigree characteristic markers.One example of this method in D ' Amour et al., NatureBiotechnology, disclosed in 2006.

For example, can be by the way that the cell for expressing endoderm pedigree characteristic markers be being contained exenatide Cultivated in culture medium, to make the cell of expression endoderm pedigree characteristic markers be further differentiated into expression pancreas The cell of gland endocrine pedigree characteristic markers.One example of this method is in D ' Amour et al., Nature Biotechnology, disclosed in 2006.

In one aspect of the invention, according to transferring LifeScan, the U.S. Patent application No. 11/736 of Inc., Method disclosed in 908, it is special by using the factor treatment expression endoderm pedigree for suppressing Notch signal transduction pathways The cell of sign property label, to make the cell of expression endoderm pedigree characteristic markers further be divided into expression pancreas The cell of gland endocrine pedigree characteristic markers.

In one aspect of the invention, according to transferring LifeScan, the U.S. Patent application No. 11/779 of Inc., Method disclosed in 311, it is special by using the factor treatment expression endoderm pedigree for suppressing Notch signal transduction pathways The cell of sign property label, to make the cell of expression endoderm pedigree characteristic markers further be divided into expression pancreas The cell of gland endocrine pedigree characteristic markers.

In one aspect of the invention, according to transferring LifeScan, the U.S. Patent application No. 60/953 of Inc., Method disclosed in 178, it is special by using the factor treatment expression endoderm pedigree for suppressing Notch signal transduction pathways The cell of sign property label, to make the cell of expression endoderm pedigree characteristic markers further be divided into expression pancreas The cell of gland endocrine pedigree characteristic markers.

In one aspect of the invention, according to U.S. Patent application No.60/990, the method disclosed in 529, passes through The cell of processing expression endoderm pedigree characteristic markers, to make expression endoderm pedigree characteristic markers Cell be further differentiated into expression pancreatic endocrine pedigree characteristic markers cell.

In one aspect of the invention, it is used to increase and the relevant mark of pancreatic endocrine pedigree the present invention provides one kind Remember the method for the expression of thing, the described method includes express pancreas with the medium treatment comprising enough TGF-β receptor stimulating agents The cell of endocrine pedigree characteristic markers, to cause the increase with the relevant marker representation of pancreatic endocrine pedigree.

The TGF-β receptor stimulating agent can be any any reagent that can be combined and activate TGF-β acceptor.At one In embodiment, the TGF-β receptor stimulating agent is selected from activin A, activin B and activin C.

In an alternative embodiment, the TGF-β receptor stimulating agent can be the peptide variation of activin A.This kind of peptide becomes The example of body, which is disclosed in, transfers Centocor R&D, the U.S. Patent application No.61/076 of Inc., in 889.

In one embodiment, express pancreatic endocrine pedigree characteristic markers cell with present in an amount at least sufficient to cause with The increased activin A of the relevant marker representation of pancreatic endocrine pedigree is handled about one to about five day.Alternatively select Select, express pancreatic endocrine pedigree characteristic markers cell with present in an amount at least sufficient to cause it is relevant with pancreatic endocrine pedigree The increased activin A of marker representation is handled about three to about five days.Alternatively, pancreatic endocrine spectrum is expressed Be characteristic markers cell with present in an amount at least sufficient to cause with the relevant marker representation of pancreatic endocrine pedigree it is increased swash Cerebroysin A is handled about five days.

In an alternative embodiment, TGF-β receptor stimulating agent is selected from GDF-8, GDF 11 and GDF-15.In a reality Apply in example, TGF-β of the cell selected from GDF-8, GDF 11 and GDF-15 of expression pancreatic endocrine pedigree characteristic markers Receptor stimulating agent and other at least one factors are handled.In one embodiment, other described at least one factors are Ring aniline-pyrido triaizine compounds.The example of this kind of ring aniline-pyrido triaizine compounds, which is disclosed in, to be transferred The U.S. Patent application No.61/076 of Centocor R&D, Inc., in 900.In an alternative embodiment, described at least one Other factors of kind are aniline-pyrido triaizine compounds.The example of this kind of aniline-pyrido triaizine compounds is disclosed in transfer The U.S. Patent application No.61/076 of Centocor R&D, Inc. are given, in 908.In an alternative embodiment, it is described at least Other a kind of factors are to be disclosed in transfer Centocor R&D, the U.S. Patent application No.61/076 of Inc., the change in 915 Compound.

In one aspect of the invention, the cell enrichment of pancreatic endocrine pedigree characteristic markers will then be expressed.It is rich Collecting step can be in the advance for the cell that expression pancreatic endocrine pedigree characteristic markers are handled with TGF-β receptor stimulating agent OK.Alternatively, enriching step can use TGF-β receptor stimulating agent processing expression pancreatic endocrine pedigree Carried out after the cell of characteristic markers.

In one embodiment, by the suspension for the cell for obtaining expression pancreatic endocrine pedigree characteristic markers And cell suspending liquid is set to pass through 40 μm of cell filtering nets to realize enrichment.

Express the detection of the cell of pancreatic endocrine pedigree characteristic markers

Pancreatic endocrine pedigree characteristic markers are well-known to those skilled in the art, and are divided in other pancreas Pedigree characteristic markers are secreted constantly to be authenticated.These labels can be used for whether determining cell processed according to the present invention It is differentiated and obtain pancreatic endocrine pedigree features characteristic.The specific marker thing of pancreatic endocrine pedigree include it is a kind of or A variety of transcription factors, such as the expression of NGN-3, NeuroD, Islet-1.

β cell lineage characteristic markers are well-known to those skilled in the art, and other β cell lineage features Property label is constantly authenticated.These labels can be used for confirming that the cell that handles according to the present invention is differentiated, to obtain β-thin The property of born of the same parents' pedigree characteristic.Inter alia, β cell lineages specific characteristics include one or more transcription factors Expression, these factors be, for example, Pdx1 (pancreas and duodenum hox genes -1), Nkx2.2, Nkx6.1, Isl1, Pax6, Pax4, NeuroD, Hnf1b, Hnf-6, Hnf-3 β and MafA and other.These transcription factors differentiate in endocrine cell leads Gain public acceptance in domain.See, for example, Edlund (Nature Reviews Genetics 3:524-632(2002)).

Can by by processed cell colony exposed to can specific recognition by expression pancreatic endocrine pedigree characteristic The reagent (such as antibody) of the protein label of the cell expression of label determines differentiation efficiency.Alternatively, Can by by processed cell colony exposed to can specific recognition by express β cell lineage characteristic markers cell The reagent (such as antibody) of the protein label of expression determines differentiation efficiency.

Method for assessing the expression of protein label and nucleic acid markers in culture or separated cell is The standard method of this area.It is in situ that these methods include quantitative reverse transcription polymerase chain reaction (RT-PCR), Northern traces Hybridization (see, for example, " Current Protocols in Molecular Biology " (Ausubel et al. (editor), 2001 Supplementary issue)) and immunoassay, such as the immunohistochemical analysis of segmentation material, western blot and for complete The flow cytometry (FACS) for the label being readily available in cell is (see, for example, " Using of Harlow and Lane Antibodies:A Laboratory Manual ", New York:Cold Spring Harbor Laboratory Press (1998))。

In one aspect of the invention, by measuring insulin positive cells in given cell culture after treatment Percentage come determine differentiation efficiency.In one embodiment, the method for the present invention produces about 100% in given culture Insulin positive cells.In an alternative embodiment, the method for the present invention produces about 90% insulin in given culture Positive cell.In an alternative embodiment, the method for the present invention produced in given culture about 80% insulin positive it is thin Born of the same parents.In an alternative embodiment, the method for the present invention produces about 70% insulin positive cells in given culture. In one alternate embodiment, the method for the present invention produces about 60% insulin positive cells in given culture.Replaced at one For in embodiment, the method for the present invention produces about 50% insulin positive cells in given culture.Substitute and implement at one In example, the method for the present invention produces about 40% insulin positive cells in given culture.In an alternative embodiment, The method of the present invention produces about 30% insulin positive cells in given culture.In an alternative embodiment, it is of the invention Method produces about 20% insulin positive cells in given culture.In an alternative embodiment, the method for the present invention exists About 10% insulin positive cells are produced in given culture.In an alternative embodiment, the method for the present invention is in given training About 5% insulin positive cells are produced in foster thing.

In one aspect of the invention, the efficiency of differentiation, pancreas are determined by measuring the insulin secretion of glucose stimulation Island element secretion can be determined by measuring the measurement of the C- peptides discharged by cell.In one embodiment, produced by the method for the present invention Cell can produce about 1000ng C- peptides/pg DNA.In an alternative embodiment, the cell produced by the method for the present invention can produce Raw about 900ng C peptides/pg DNA.In an alternative embodiment, the cell produced by the method for the present invention can produce about 800ng C peptides/pg DNA.In an alternative embodiment, the cell produced by the method for the present invention can produce about 700ng C peptides/pg DNA.In an alternative embodiment, the cell produced by the method for the present invention can produce about 600ng C peptides/pg DNA.One In a alternate embodiment, the cell produced by the method for the present invention can produce about 500ng C peptides/pg DNA.Substitute and implement at one In example, the cell produced by the method for the present invention can produce about 400ng C peptides/pg DNA.In an alternative embodiment, by this The cell that inventive method produces can produce about 500ng C peptides/pg DNA.In an alternative embodiment, produced by the method for the present invention Raw cell can produce about 400ng C peptides/pg DNA.In an alternative embodiment, the cell produced by the method for the present invention can Produce about 300ng C peptides/pg DNA.In an alternative embodiment, the cell produced by the method for the present invention can produce about 200ng C peptides/pg DNA.In an alternative embodiment, by the method for the present invention produce cell can produce about 100ng C peptides/ pg DNA.In an alternative embodiment, the cell produced by the method for the present invention can produce about 90ng C peptides/pg DNA.One In a alternate embodiment, the cell produced by the method for the present invention can produce about 80ng C peptides/pg DNA.Substitute and implement at one In example, the cell produced by the method for the present invention can produce about 70ng C peptides/pg DNA.In an alternative embodiment, by this hair The cell that bright method produces can produce about 60ng C peptides/pg DNA.In an alternative embodiment, produced by the method for the present invention Cell can produce about 50ng C peptides/pg DNA.In an alternative embodiment, the cell produced by the method for the present invention can produce Raw about 40ng C peptides/pg DNA.In an alternative embodiment, the cell produced by the method for the present invention can produce about 30ng C Peptide/pg DNA.In an alternative embodiment, the cell produced by the method for the present invention can produce about 20ng C peptides/pg DNA. In an alternative embodiment, the cell produced by the method for the present invention can produce about 10ng C peptides/pg DNA.

Therapy

In one aspect, it is used to treat the present invention provides one kind and suffers from type 1 diabetes, or has 1 patients with type Ⅰ DM wind of development The method of the patient of danger.This method, which is related to, cultivates multipotential stem cell, makes the multipotential stem cell vitro differentiation into beta cell Pedigree, and by the cell transplantation of the beta cell pedigree into patient.

In another aspect, it is used to treat the present invention provides one kind and suffers from diabetes B, or has development diabetes B The method of the patient of risk.This method, which is related to, is cultivated multipotential stem cell, the cells in vitro of the culture is divided into β-thin Born of the same parents' pedigree, and by the cell transplantation of the beta cell pedigree into the patient.

If appropriate, patient can be carried out with the medicament or bioactivator for being conducive to the implantation cell survival and function Further processing.These reagents may include (such as) insulin, member's (including TGF-β 1,2 and 3) of TGF-β family, bone shape It is small that albumen (BMP-2, -3, -4, -5, -6, -7, -11, -12 and -13), fibroblast growth factor -1 and -2, blood occur for state Plate derivative growth factor-AA and-BB, platelet rich plasma, insulin-like growth factor (IGF-I, II), growth and differentiation factor (GDF-5, -6, -7, -8, -10, -15), vascular endothelial cell derivative growth factor (VEGF), multiple effect protein (pleiotrophin), Endothelin etc..Other drugs compound may include (such as) niacinamide, glucagon-like peptide-I (GLP-1) and glucagon-like peptide-II, GLP-1 and 2 analogue bodies, exendin-4, retinoic acid, parathyroid hormone, For example US published patent application 2004/0209901 of MAPK inhibitor and US published patent application 2004/ Compound disclosed in 0132729.

Multipotential stem cell can be made to be divided into insulin producing cell before transplanting into recipient.In a specific implementation In example, transplanting into before recipient, multipotential stem cell is divided into beta cell completely.Alternatively, can be by multipotency Stem cell is with the state migration of undifferentiated state or partial differentiation into recipient.It can occur further to divide in the recipient Change.

Can by definitive endodenn cells or, alternately, endoderm cells, or alternatively, β Cell is transplanted as cell dispersion, or these cells can be formed cluster, can be transfused these cell clusters into vena portae hepatica It is interior.Alternatively, cell can be arranged in the degradable polymer type holder of biocompatibility, be porous non- In degradability device or it can be packaged to protected from the destruction of host immune response.Can be by cell transplantation into receiving In suitable position in person.Implantation site include (such as) liver, original pancreas, subrenal capsule space, nethike embrane, peritonaeum, slurry Film down space, intestines, stomach or subcutaneous pocket.

In order to strengthen further differentiation, survival rate or the activity of implantation cell, can before dosed cells, simultaneously Or the extra factor is applied afterwards, such as growth factor, antioxidant or antiinflammatory.In certain embodiments, growth factor is used In the cell that differentiation in vivo is applied.These factors can be secreted by endogenous cell and on the spot exposed to the cell applied.It can pass through Any combination for the growth factor that endogenous growth factor known in the art or external source are applied induces the implantation cell to be divided Change.

Amount used depends on many factors, including the situation of patient and the response to the therapy in transplanting, and can be by Those skilled in the art determine.

In one aspect, it is used to treat the present invention provides one kind and suffers from diabetes, or has the trouble of development diabetes risk The method of person.This method is related to culture multipotential stem cell, the cells in vitro of the culture is divided into beta cell pedigree, and should In the three-dimensional holder of cell incorporation.Transplanting into before patient, which can maintained in vitro on the holder.As another One kind selection, can by the holder directly transplanting comprising the cell into patient without carrying out extra in vitro culture.It can appoint Choosing mixes the medicament of at least one survival for being conducive to implantation cell and function in the holder.

Suitable for the object of the invention support material include available for the tissue templates of tissue repair, conduit, barrier and Storage.Specifically, it has been used to biological tissue in vitro and in vivo rebuild or regenerate, and has lured for delivering chemoattractant Lead the synthetic material and natural material of the foam of tissue growth, sponge, gel, hydrogel, yarn fabric and non-woven structure form Suitable for the method for the practice present invention.See, e.g. special in United States Patent (USP) 5,770,417, United States Patent (USP) 6,022,743, the U.S. Profit 5,567,612, United States Patent (USP) 5,759,830, United States Patent (USP) 6,626,950, United States Patent (USP) 6,534,084, United States Patent (USP) 6, 306,424th, United States Patent (USP) 6,365,149, United States Patent (USP) 6,599,323, United States Patent (USP) 6,656,488, US published Material disclosed in patent application 2004/0062753A1, United States Patent (USP) 4,557,264 and United States Patent (USP) 6,333,029.

In order to form the supporting mass containing medicament, medicament can be mixed with polymer solution before supporting mass is formed.As Medicament, can be coated on the holder made, be carried out preferably in the case of there are pharmaceutical carrier by another kind selection.Medicine Can exist with liquid, finely-divided solid or any other suitable physical form.Alternatively, excipient can be added To change the rate of release of medicament in holder.In an alternative embodiment, by least one medicine for anti-inflammatory compound In compound incorporation holder, such as in United States Patent (USP) 6, the compound disclosed in 509,369.

At least one medical compounds for anti-apoptotic compound can be mixed in holder, such as in United States Patent (USP) 6, Compound disclosed in 793,945.

At least one medical compounds for fibre modification inhibitor can be mixed in holder, such as in United States Patent (USP) Compound disclosed in 6,331,298.

Holder can also be such as US published mixed with least one medical compounds that can strengthen angiogenesis Compound disclosed in patent application 2004/0220393 and US published patent application 2004/0209901.

Holder can also be such as US published mixed with least one medical compounds for immunosuppressive compounds Compound disclosed in patent application 2004/0171623.

Holder can also be mixed with member's (bag of at least one medical compounds for growth factor, such as TGF-β family Include TGF-β 1,2 and 3), bone morphogenetic protein (BMP-2, -3, -4, -5, -6, -7, -11, -12 and -13), fibroblast Growth factor-1 and -2, platelet-derived growth factor-AA and-BB, platelet rich plasma, insulin-like growth factor (IGF- I, II), growth and differentiation factor (GDF-5, -6, -8, -10, -15), vascular endothelial cell derivative growth factor (VEGF), multiple-effect Albumen, Endothelin etc..Other drugs compound may include (such as) niacinamide, Hypoxia inducible factor-lα, hyperglycemic factor Sample peptide-I (GLP-1), GLP-1 and GLP-2 analogue bodies, exendin-4, nodal, noggin, NGF, retinoic acid, first shape Other glandular hormone, tenascin-C, tropoelastin, the peptide of thrombin derivative, cathelicidin (cathelicidin), defence Plain (defensin), laminin, contain adherent cell extracellular matrix protein such as fibronectin and vitronectin The biological peptide of cell-binding domain and heparin binding domain, MAPK inhibitor are (such as in US published patent application 2004/0209901 and US published patent application 2004/0132729 disclosed in compound).

Cell of the present invention can be mixed in stent to realize by the way that cell is simply deposited on the stent.Cell can (J.Pediatr.Surg.23 (1 Pt 2) in stent is entered by simple diffusion:3-9(1988)).Have been developed it is some its His method strengthens the efficiency of cell inoculation.For example, rolling bottle is used to cartilage cell being inoculated in polyglycolic acid stent (Biotechnol.Prog.14(2):193-202(1998)).It is to utilize centrifugation for cell inoculation another kind method, it is this Centrifugation produces minimum stress to the cell of inoculation and strengthens inoculation efficiency.For example, Yang et al. has developed a kind of cell inoculation Method (J.Biomed.Mater.Res. 55 (3):379-86 (2001)), it is known as centrifuge cell and fixes (Centrifugational Cell Immobilization, CCI).

The present invention is further illustrated by (but not limited to) following instance.

Example

Example 1

Differentiation of the human embryo stem cell of cell line H1 to pancreatic endocrine cell in the case of there is no hyclone

By the human embryonic stem cell H1 cells in the 52nd generation coated with MATRIGELTMThe culture dish of (1: 30 dilution) It is upper to cultivate and exposed to being supplemented with 2%BSA (catalog number (Cat.No.) 152401, MP Biomedical, Ohio) and 100ng/ml activin As (R&D Systems, MN)+20ng/ml WNT-3a (catalog number (Cat.No.) 1324-WN-002, R&D Systems, MN)+8ng/ml's The RPMI culture mediums of bFGF (catalog number (Cat.No.) 100-18B, PeproTech, NJ) one day, then with being supplemented with 2%BSA and 100ng/ Ml activin As add the RPMI culture mediums of 8ng/ml bFGF to reprocess two days.Then, by culture DMEM/F12+2%BSA+ 50ng/ml FGF7+0.25 μM cyclopamines-KAAD (#239804, Calbiochem, CA) are handled two days, then in DMEM/F12 + 1%B27 (Invitrogen, CA)+50ng/ml FGF7+0.25 μM-KAAD+2 μM of retinoic acids of cyclopamine (RA) (Sigma, MO) incubated four days in+100ng/ml noggins (R&D Systems, MN).

By by cell in+1 μM of DAPT of DMEM/F12+1%B27 (Invitrogen, CA)+100ng/ml noggins (inhibitors of gamma-secretase) (catalog number (Cat.No.) 565784, Calbiochem, CA)+1 μM of ALK5 inhibitor II (catalog number (Cat.No.) 616452, Calbiochem, Ca)+100ng/ml leads in protein-4 (R&D Systems, MN) and incubates three days, then in DMEM/F12+1% Incubated again seven days in+1 μM of ALK5 inhibitor II (Calbiochem, Ca) of B27 (Invitrogen, CA), to make cell differentiation For pancreatic endocrine cell.The last stage is added further to make endocrine culture ripe, the stage by Processing is formed for seven days in DMEM/F12+1%B27 (Invitrogen, CA).In addition to the final stage, the every other stage Include daily replacement culture medium.Fig. 1 schematically illustrates this method.In each stage, cell number is calculated simultaneously with hemocytometer RNA is collected to analyze for PCR.All samples are collected in triplicate.

The component a and component b of Fig. 2 shows being surveyed by FACS on three days (3D) this time points corresponding to the 1st stage The data of CXCR4 expression and real-time PCR in the cell obtained.The change multiple of expression is done carefully relative to undifferentiated H1 embryos Born of the same parents show.

Example 2

The expression of pancreatic endocrine label is significantly improved to the 6th stage culture addition activin A

By the cell of the human embryonic stem cell H1 in the 43rd generation coated with MATRIGELTMThe culture of (1: 30 dilution) Cultivated on ware, and the method according to example 1 is divided into pancreatic endocrine cell.In the 6th stage, some cultures are used The activin A (R&D Systems, MN) of 10ng/ml is handled seven days.At the 7th day, cell is used into 1X Accutase at room temperature (Sigma, MO) is handled 5 minutes, is then removed Accutase and is added DMEM/12+1%B27.By attached cell cell scraper Removal and lightly settling flux, and be passed to 40 μm of cell filtering nets.Liquid stream is by the way that removal stays in thin on filter screen Born of the same parents, collect sample and are dyed for PCR and dithizone.Dithizone (DTZ) is the dyestuff of selective binding zinc, it has proved that pancreas There are zinc in island.Fig. 3 (component a-c) shows the training of the DTZ dyeing before and after using 40 μm of cell filtering net separation Support thing.Most of (about 80%) is more than 40 μm of cell cluster DTZ stained positives.This provide it is a kind of rapidly and simply The enrichment method of endocrine enrichment of cell cluster.Fig. 4 (component a-f) shows the activin A with +/- 10ng/ml in the 6th stage The gene expression profile for the cell for handling and being then enriched with using 40 μm of filters.Activin A was added to for the 6th stage and significantly improves The expression of all crucial pancreatic endocrine labels.

Thereby the publication quoted in entire chapter document is incorporated by reference being incorporated herein.Although combine above real Example and the preferred embodiment various aspects that the invention has been described, it is to be understood that the scope of the present invention is from above-mentioned specific implementation The restriction of mode, and limited by the claims correctly explained under Patent Law principle below.

Claims (4)

1. a kind of method increased with the expression of the relevant label of pancreatic endocrine pedigree, the described method includes sufficient with amount is included To cause the culture medium with the increased activin A of the relevant marker representation of pancreatic endocrine pedigree, handle in expression and pancreas Secrete the cell of the relevant label of pedigree one to five day.
2. according to the method described in claim 1, wherein by obtaining the thin of expression pancreatic endocrine pedigree characteristic markers The suspension of born of the same parents, and cell suspending liquid is separated by filter screen, by the expression and the relevant mark of pancreatic endocrine pedigree The cell of note thing is enriched with;The cell of wherein described expression endocrine marker characteristic markers is protected by filter screen Hold.
3. a kind of method for making pluripotent stem cell differentiation, described method includes following steps:
A) multipotential stem cell is cultivated,
B) make the pluripotent stem cell differentiation into expression definitive entoderm pedigree characteristic markers cell,
C) cell differentiation for expressing definitive entoderm pedigree characteristic markers is made into expression endoderm pedigree feature The cell of property label,
D) cell differentiation for expressing endoderm pedigree characteristic markers is made into expression pancreatic endocrine pedigree feature The cell of property label;And
E) it is enough to cause the culture medium with the increased activin A of the relevant marker representation of pancreatic endocrine pedigree with comprising amount, Handle the cell of the expression pancreatic endocrine pedigree characteristic markers one to five day,
Wherein described multipotential stem cell is selected from human embryonic stem cell H1, H7, H9 and BG01v.
4. according to the method described in claim 3, wherein by obtaining the thin of expression pancreatic endocrine pedigree characteristic markers The suspension of born of the same parents, and cell suspending liquid is separated by filter screen, by the expression and the relevant mark of pancreatic endocrine pedigree The cell of note thing is enriched with;The cell of wherein described expression endocrine marker characteristic markers is protected by filter screen Hold.
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